Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited disorder that manifests primarily as a neuromuscular disease but can also involve cardiac, endocrine, gastrointestinal, and central nervous system dysfunction. A hallmark symptom of DM1 is myotonia, a defect in muscle relaxation following contraction and detected by electromyography (EMG) as long runs of repetitive action potentials in muscle. The genetic lesion in DM1 consists of an unstable CUG trinucleotide repeat element in the 3′ untranslated region of a gene encoding a protein kinase known as dystrophia myotonica-protein kinase (DMPK). A small number of copies of the CUG repeat are found in unaffected individuals, whereas large numbers of unstable CUG repeats, ranging from 50 copies to several thousand copies, are detected in DM1 patients. Genetic studies have established that the number of CUG repeats correlates directly with the age of onset and disease severity (Day & Ranum, 2005, Neuromuscul Disord 15:5-16).
RNA transcripts such as DMPK that contain a large number of CUG repeats acquire gain-of-function properties and promote RNA-mediated toxicity in cells and tissues expressing the mutant DMPK transcript (Wheeler &Thornton, 2007, Curr Opin Neurol 20:572-576). The pathogenic transcript is retained in the nucleus (Taneja et al., 1995, J Cell Biol 128:995-1002) where it entraps RNA binding proteins such as MBNL1 (Mankodi et al., 2001, Hum Mol Genet 10:2165-2170; Mankodi et al., 2003, Ann Neurol 54: 760-768). Poly (CUG) RNA also increases steady-state levels of CUG-BP1 (Timchenko NA et al., 2001, J Biol Chem, 276:7820-7826; Kuyumcu-Martinez et al., 2007, Mol Cell 28:68-78). Both sequestration of MBNL1 and an increase in CUG-BP 1 activity is associated with abnormal splicing of a large number of RNA transcripts. Of note is the defective RNA splicing of chloride channel C1C-1 mRNA (Mankodi et al., 2002, Mol Cell 10:35-44), which has been shown to directly result in myotonia (Wheeler et al., 2007, J Clin Invest 117:3952-3957). In addition to abnormal RNA splicing, a consequence of mutant DMPK expression includes a remodeling of the skeletal muscle transcriptome (Osborne et al., 2009, Hum Mol Genet 18:1471-1481). Protein products with altered primary sequences resulting from abnormal RNA splicing and the dysregulated mRNA levels comprising the altered transcriptome would be expected to have associations and even causal relationships with specific aspects of DM1 disease.
There are currently no therapeutic agents in the clinic for DM1 that target toxic RNA, the primary pathogenic driver of the disease. Standard-of-care therapies for DM1 are mainly supportive and aimed at managing specific symptoms, e.g. myotonia (Logigian et al., 2010, Neurology 74:1441-1448). Pre-clinical evaluation of novel therapeutic approaches have been conducted in the HSALR transgenic mouse model that contains a human skeletal actin transgene harboring a 250 CUG trinucleotide (SEQ ID NO: 14) insertion in the 3′ untranslated region. The HSALR model demonstrates several features of DM1including MBNL1sequestration by CUG RNA and the ensuing RNA splicing abnormalities, alterations in the muscle transcriptome, and physiological aberrations such as myotonia (Osborne et al., 2009, Hum Mol Genet 18:1471-1481; Mankodi et al., (2000), Science 289:1769-1773). Novel therapeutic modalities tested in the HSALR mouse include small molecule ligands that are designed to interact with CUG repeat RNA and liberate foci-associated MBNL1 protein (Warf et al., 2009, Proc Natl Acad Sci USA 106:18551-18556; Parkesh et al., 2012, J Am Chem Soc 134:4731-4742; Ofori et al., 2012, Nucl. Acid. Res. in press, first published online: Apr. 6, 2012). In addition, three antisense oligonucleotide (ASO) chemistries that target the CUG repeat tract have been assessed in studies conducted in transgenic mouse models of DM1(Wheeler et al., 2009, Science 325:336-339; Mulders et al., 2009, Proc Natl Acad Sci U S A 106:13915-13920; Lee et al., 2012, Proc Natl Acad Sci USA 109:4221-4226. Wheeler and colleagues (2009, Science 325:336-339) demonstrated local correction of DM1 pathology in tibilias anterior (TA) muscle of HSALR mice subjected to intramuscular (IM) injection of a 25 mer morpholino oligonucleotide of CAG sequence (CAG25). The CAG25-treated TA muscles showed a decrease in the presence of ribonuclear foci, re-distribution of MBNL1 protein, correction of abnormal RNA splicing, restoration of chloride channel (CIC-1) protein expression and function, and reduction of myotonia (Wheeler et al., 2009, Science 325:336-339). Although this therapeutic approach showed corrections in the DM1-like phenotype of HSALR mice, thereremains a need for a systemic delivery strategy that would allow multiple tissues, including multiple tissue types, to gain exposure to active morpholino oligonucleotide targeted at the toxic DMPK RNA transcripts responsible for DM1.
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